Bioprocessing

ABSTRACT

Bioreactors are provided that include a vessel and a jet mixer disposed in the vessel. Methods that utilize the bioreactors are provided, involving placing a microorganism or cells and a fluid medium in the bioreactor.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/592,571, filedMay 11, 2017, which is a continuation of U.S. Ser. No. 15/044,013, filedFeb. 15, 2016, now U.S. Pat. No. 9,677,043, issued on Jun. 13, 2017,which is a continuation of U.S. Ser. No. 14/196,589, filed Mar. 4, 2014,now U.S. Pat. No. 9,284,586, issued Mar. 15, 2016, which is acontinuation of U.S. Ser. No. 13/293,977, filed Nov. 10, 2011, now U.S.Pat. No. 8,669,099, issued Mar. 11, 2014, which is a continuation ofPCT/US2010/035328, filed May 18, 2010, which claimed priority to U.S.Provisional Application Ser. No. 61/179,995, filed May 20, 2009, U.S.Provisional Application Ser. No. 61/218,832, filed Jun. 19, 2009, andU.S. Provisional Application Ser. No. 61/218,803, filed Jun. 19, 2009.The complete disclosure of each of these applications is herebyincorporated by reference herein.

BACKGROUND

Bioreactors, often referred to as fermentors, are commonly used inbioprocessing. “Bioprocessing” refers to aerobic and anaerobic processesthat involve microorganisms, e.g., cells, in a medium. Examples ofbioprocesses include yeast fermentation, bacterial fermentation, cellculture, bacterial culture, and the production of a product using cells,e.g., using mammalian cells such as CHO cells to express a protein,e.g., a therapeutic protein, or an enzyme.

A bioreactor generally includes a vessel in which a bioprocess iscarried out, and sensors and process controls that allow parameters ofthe process to be monitored and controlled. Bioreactors also typicallyinclude an agitator, for example a Rushton or marine impeller, thatmixes the vessel contents during bioprocessing.

It is generally important to carefully control process parameters duringbioprocessing, for example gas flow rates, temperature, pH, dissolvedoxygen level, and agitation speed and conditions. Dissolved oxygen levelis a measure of oxygen transfer from gas to liquid phase, which isimportant to many bioprocesses and can be difficult to accomplish. Whileoxygen transfer is generally helped by agitation, agitation speed isoften limited by power consumption and in some cases the risk of damageto the microorganisms. In some cases, for example, in the case ofmammalian cells, the microorganisms are fragile and may be sensitive toheat, shear, and/or other process conditions.

SUMMARY

Generally, this invention relates to bioreactors, and to bioprocessingmethods using bioreactors.

Bioprocessing can be enhanced by the use of certain mixing techniquesand equipment, which may enhance the reaction rate and improve theefficiency of the process. The mixing techniques and equipment disclosedherein also enhance mass transfer, and as a result reaction rates in amixture, and avoid or minimize harm to sensitive ingredients of themixture such as microorganisms and enzymes. In particular, jet mixingtechniques, including for example jet aeration and jet flow agitation,have been found to provide good wetting, dispersion and mechanicaldisruption. The mixing techniques and equipment disclosed herein can insome cases allow the solids level of the mixture being processed to beincreased, and thus the process can proceed more rapidly, moreefficiently and more cost-effectively, and the resulting concentrationof the final product can be increased. The mixing systems describedherein generally do not impart high shear to the liquid. In someimplementations, the mixing systems do not cause a deleterious increasein the overall temperature of the liquid. As a result, microorganismsand cells used in bioprocessing are maintained in a viable conditionthroughout the process.

In one aspect, the invention features a bioreactor that includes avessel and a jet mixer disposed in the vessel. In another aspect, theinvention features a method that includes placing a microorganism orcells and a fluid medium in a vessel, and mixing the contents of thevessel by operation of a jet mixer.

Some embodiments include one or more of the following features. Thebioreactor can further include sensors and process controllers. Forexample, the bioreactor can further include a temperature control systemconfigured to regulate the temperature in the vessel. The bioreactor caninclude a gas delivery system configured to supply a process gas, e.g.,air, oxygen, nitrogen or carbon dioxide, to the vessel. In some casesthe vessel includes a vent, and the bioreactor includes a source ofoxygen in communication with the vessel, an oxygen monitor configured tomonitor the oxygen content of a liquid in the vessel, an a controllerconfigured to adjust the oxygen content of the liquid, using the ventand oxygen source, in response to input from the oxygen monitor. Theflow rate of oxygen into the vessel, if oxygenation is required, can berelatively low. For example, the controller may be configured tooxygenate the vessel at a rate of less than 0.2 vvm, e.g., less than0.1, 0.05, 0.025, or even less than 0.01 vvm.

The bioreactor may further include a concentration monitor configured tomonitor the concentration of a product in the liquid in the vessel, anda controller configured to stop the bioprocess based on input receivedfrom the concentration monitor. In some cases, the system includes aprocess-terminating module configured to stop the bioprocess in responseto a signal received from the controller.

The jet mixer may include, for example, a jet-flow agitator, a jetaeration type mixer, or a suction chamber jet mixer. If a jet aerationtype mixer is used, it may be used with or without injection of airthrough the mixer. For example, if the jet aeration type mixer includesa nozzle having a first inlet line and a second inlet line, in somecases both inlet lines are supplied with a liquid.

The bioreactor may include delivery devices configured to deliver themedium, microorganisms or cells, and/or any other materials used in thebioprocess, such as buffers, foam control agents, and the like, to thevessel. The bioreactor may include an outlet through which effluent canbe collected. The jet mixer can further include a motor, and thebioreactor can further include a device configured to monitor the torqueon the motor during mixing and adjust the operation of the deliverydevices and/or the outlet, and/or the torque of the motor, based oninput from the torque-monitoring device. In addition, or alternatively,the bioreactor may include an amp monitoring device, configured tomeasure the full-load amperage of the motor. The bioreactor may alsoinclude a variable frequency drive (VFD) configured to adjust the speedof the motor.

In some embodiments, the mixer is configured to limit any increase inthe overall temperature of a fluid medium in the vessel to less than 5degrees C. over the course of mixing.

In another aspect, the invention features a bioreactor that includes amixer that produces generally toroidal flow within the vessel. Theinvention also features methods of bioproces sing using the bioreactor.These aspects of the invention may include, in some embodiments, any oneor more of the features discussed above.

Examples of products that can be produced by employing the bioreactorsand methods described herein include hydrocarbons; proteins (e.g.,therapeutic proteins); enzymes (e.g., cellulolytic enzymes); alcohols(e.g., monohydric alcohols or dihydric alcohols, such as ethanol,n-propanol or n-butanol); xylitol; carboxylic acids, such as acetic acidor butyric acid; salts and esters of carboxylic acids; mixtures ofcarboxylic acids with salts and/or esters of carboxylic acids (e.g.,methyl, ethyl and n-propyl esters); ketones (e.g., acetone); aldehydes(e.g., acetaldehyde); alpha and beta unsaturated acids, such as acrylicacid; and olefins, such as ethylene. Specific examples include ethanol,propanol, propylene glycol, butanol, 1,4-butanediol, 1,3-propanediol,methyl or ethyl esters of any of these alcohols, methyl acrylate,methylmethacrylate, lactic acid, proprionic acid, butyric acid, succinicacid, 3-hydroxypropionic acid, salts of any of these acids, and mixturesof any of these acids and their respective salts. These and otherproducts are described in U.S. Ser. No. 12/417,900, the disclosure ofwhich is incorporated by reference herein.

All publications, patent applications, patents, and other referencesmentioned herein or attached hereto are incorporated by reference intheir entirety for all that they contain.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a bioreactor according to one embodiment.

FIGS. 2 and 2A are diagrams illustrating jet flow exiting a nozzle.

FIG. 3 is a diagrammatic perspective view of a jet-flow agitatoraccording to one embodiment. FIG. 3A is an enlarged perspective view ofthe impeller and jet tube of the jet-flow agitator of FIG. 3. FIG. 3B isan enlarged perspective view of an alternate impeller.

FIG. 4 is a diagram of a suction chamber jet mixing nozzle according toone embodiment.

FIG. 5 is a diagrammatic perspective view of a jet mixing nozzle for asuction chamber jet mixing system according to another embodiment.

FIG. 6 is a side view of a bioreactor including a jet aeration typesystem according to one embodiment, showing a multi-level arrangement ofnozzles in a vessel.

FIG. 7 is a perspective view of a jet aeration mixer according to oneembodiment.

FIG. 8 is a cross-sectional view of a jet aeration type mixer accordingto one embodiment.

FIG. 9 is a cross-sectional view of a jet aeration mixer according to analternate embodiment.

FIGS. 10 and 10A are perspective views of two embodiments of mixingheads used in a mixer according to an alternate embodiment.

FIG. 11 is a cross-sectional view of a vessel having a domed bottom andtwo jet mixers extending into the vessel from above.

DETAILED DESCRIPTION

Referring to FIG. 1, a bioreactor 100 includes a vessel 102 in which abioprocess takes place. Vessel 102 is generally of an autoclavable,inert material such as glass or stainless steel, and may or may not bejacketed. In some cases, the vessel may be relatively low volume, e.g.,less than about 30,000 L. In other cases, the vessel may be relativelyhigh volume, e.g., greater than 30,000 L, such as from about 50,000 L to2 million liters, or even greater than 2 million liters. Suitable lowvolume vessels may have a total capacity, for example, of from about 5 Lto 30,000 L, e.g., 50 L to 5000 L. For example, the total capacity ofthe vessel may be 75 L, 150 L, 300 L, 500 L, 1000 L, 1500 L, 3000 L, or5000 L. It may be preferred that the vessel have an aspect ratio(diameter:height) of, for example, about 0.5:1 to about 4:1, or about0.5:1 to 2:1, e.g., about 0.5:1 to 1:1.

A shaft 104 extends into the vessel, and a jet mixing device 106 ismounted at the distal end of the shaft. As will be discussed in detailbelow, the jet mixing device 106 creates a circulating flow in theliquid 108 in the vessel, as indicated by the arrows entering andleaving the jet mixing device. In the embodiment shown in FIG. 1, thejet mixing device 106 includes a shroud 110 and an impeller 112 disposedwithin the shroud. However, the jet mixer may have any of the structuresdisclosed herein, or any other desired structure which produces jetflow. In the embodiment shown in FIG. 1, the shaft 104 is driven by amotor 114. However, other types of jet systems may be used as will bediscussed below.

The bioreactor may include any other desired bioreactor components andfeatures. For example, as shown in FIG. 1, bioreactor 100 includes pumps116, a container 118 for base solution, a container 120 for feed medium,a collection reservoir 122, and a source of gas 124 (multiple gassources and appropriate valving and mixing may be included if desired).Air flow control is provided by a thermal mass flow controller 125. Thebioreactor 100 also includes a dissolved oxygen probe 126, a pH probe128, and a redox (ORP) probe 130 for monitoring these parameters, and asampling probe 131 for sampling or harvesting the vessel contents. Acondenser 132 is provided for condensation of volatiles.

The bioreactor generally includes a controller (not shown) that isconfigured to control the process parameters by receiving signals fromvarious monitoring devices (e.g., the probes shown in FIG. 1) andadjusting the process parameters based on this data. For example, inbioreactor 100 the controller is configured to receive signals from theoxygen probe 126, pH probe 128, and redox probe 130, and control theinput of gas, base solution, and feed solution, respectively, based onthese signals. The controller may be, for example, a programmable logiccontroller (PLC) with an operator interface. The controller mayoptionally be configured to control multiple vessels.

In some implementations, gas delivery is through the shaft of the jetmixing device. For example, the shaft 104 may include a bore throughwhich gas is delivered, and one or more orifices through which gas exitsinto the vessel. The orifices may be within the shroud 110, to enhancemixing, and/or at other locations along the length of shaft 104.

The bioreactor may also include a number of other features to enhancebioprocessing. For example, the vessel may be cooled, e.g., with a waterjacket or other cooling system, to maintain a desired processtemperature. A foam detector may be provided, and an anti-foam deliverysystem may be included to deliver an anti-foam agent to the vesselshould excessive foaming occur. In some implementations, the bioreactoris sterilizable in place, e.g., with an automatic steam injectionsystem. Various access ports may be provided in the vessel.

Mixing Characteristics

Various types of mixing devices are described below, and other mixingdevices may be used. Suitable mixers have in common that they producehigh velocity circulating flow, for example flow in a toroidal orelliptical pattern. Generally, preferred mixers exhibit a high bulk flowrate. Preferred mixers provide this mixing action with relatively lowenergy consumption. It is also generally preferred that the mixerproduce relatively low shear and avoid heating of the liquid medium, asshear and/or heat can deleteriously affect the microorganism or cells.As will be discussed in detail below, some preferred mixers draw themixture through an inlet into a mixing element, which may include arotor or impeller, and then expel the mixture from the mixing elementthrough an outlet nozzle. This circulating action, and the high velocityof the jet exiting the nozzle, assist in dispersing material that isfloating on the surface of the liquid or material that has settled tothe bottom of the vessel, depending on the orientation of the mixingelement. Mixing elements can be positioned in different orientations todisperse both floating and settling material, and the orientation of themixing elements can in some cases be adjustable.

For example, in some preferred mixing systems the velocity v_(o) of thejet as it meets the ambient fluid is from about 2 to 300 m/s, e.g.,about 5 to 150 m/s or about 10 to 100 m/s. The power consumption of themixing system may be about 20 to 1000 KW, e.g., 30 to 570 KW, 50 to 500KW, or 150 to 250 KW for a 100,000 L tank. It is generally preferredthat the power usage be low for cost-effectiveness.

Jet Mixing

Jet mixing involves the discharge of a submerged jet, or a number ofsubmerged jets, of high velocity liquid into a fluid medium, in thiscase the mixture in the vessel of the bioreactor. The jet of liquidpenetrates the fluid medium, with its energy being dissipated byturbulence and some initial heat. This turbulence is associated withvelocity gradients (fluid shear). The surrounding fluid is acceleratedand entrained into the jet flow, with this secondary entrained flowincreasing as the distance from the jet nozzle increases. The momentumof the secondary flow remains generally constant as the jet expands, aslong as the flow does not hit a wall, floor or other obstacle. Thelonger the flow continues before it hits any obstacle, the more liquidis entrained into the secondary flow, increasing the bulk flow in thevessel. When it encounters an obstacle, the secondary flow will losemomentum, more or less depending on the geometry of the vessel, e.g.,the angle at which the flow impinges on the obstacle. It is generallydesirable to orient the jets and/or design the vessel so that hydrauliclosses to the vessel walls are minimized. For example, it may bedesirable for the vessel to have an arcuate bottom (e.g., a domedheadplate), and for the jet mixers to be oriented relatively close tothe sidewalls, as shown in FIG. 11. The vessel bottom may have anydesired domed configuration, or may have an elliptical or conicalgeometry.

Jet mixing differs from most types of liquid/liquid and liquid/solidmixing in that the driving force is hydraulic rather than mechanical.Instead of shearing fluid and propelling it around the mixing vessel, asa mechanical agitator does, a jet mixer forces fluid through one or morenozzles within the vessel, creating high-velocity jets that entrainother fluid. The result is shear (fluid against fluid) and circulation,which mix the vessel contents efficiently.

Referring to FIG. 2, the high velocity gradient between the core flowfrom a submerged jet and the surrounding fluid causes eddies. FIG. 2Aillustrates the general characteristics of a submerged jet. As thesubmerged jet expands into the surrounding ambient environment thevelocity profile flattens as the distance (x) from the nozzle increases.Also, the velocity gradient dv/dr changes with r (the distance from thecenterline of the jet) at a given distance x, such that eddies arecreated which define the mixing zone (the conical expansion from thenozzle).

In an experimental study of a submerged jet in air (the results of whichare applicable to any fluid, including water), Albertson et al.(“Diffusion of Submerged Jets,” Paper 2409, Amer. Soc. of CivilEngineers Transactions, Vol. 115:639-697, 1950, at p. 657) developeddimensionless relationships for v(x)_(r=0)/v_(o) (centerline velocity),v(r)_(x)/v(x)_(r=0) (velocity profile at a given x), Q_(x)/Q_(o) (flowentrainment), and E_(x)/E_(o) (energy change with x):

(1) Centerline velocity, v(x)_(r=0)/v_(o):

${\frac{v\left( {r = 0} \right)}{v_{o}}\frac{x}{D_{o}}} = 6.2$

(2) velocity profile at any x, v(r)_(x)/v(x)_(r=0):

${\log \left\lbrack {\frac{{v(r)}_{x}}{v_{o}}\frac{x}{D}} \right\rbrack} = {0.79 - {33\frac{r^{2}}{x^{2}}}}$

(3) Flow and energy at any x:

$\begin{matrix}{\frac{Q_{x}}{Q_{o}} = {0.32\frac{x}{D_{o}}}} & (10.21) \\{\frac{E_{x}}{E_{o}} = {4.1\frac{D_{o}}{x}}} & (10.22)\end{matrix}$

where:v(r=0)=centerline velocity of submerged jet (m/s),v_(o)=velocity of jet as it emerges from the nozzle (m/s),x=distance from nozzle (m),r=distance from centerline of jet (m),D_(o)=diameter of nozzle (m),Q_(x)=flow of fluid across any given plane at distance x from the nozzle(m³/s),Q_(o)=flow of fluid emerging from the nozzle (m³/s),E=energy flux of fluid across any given plane at distance x from thenozzle (m³/s),E_(o)=energy flux of fluid emerging from the nozzle (m³/s).

(“Water Treatment Unit Processes: Physical and Chemical,” David W.Hendricks, CRC Press 2006, p. 411.)

Jet mixing is particularly cost-effective in large-volume (over 1,000gal) and low-viscosity (under 1,000 cPs) applications. It is alsogenerally advantageous that in most cases the pump or motor of the jetmixer not be submerged, e.g., when a pump is used it is generallylocated outside the vessel.

One advantage of jet mixing is that the temperature of the ambient fluid(other than directly adjacent the exit of the nozzle, where there may besome localized heating) is increased only slightly if at all. Forexample, the temperature may be increased by less than 5° C., less than1° C., or not to any measurable extent.

Jet-Flow Agitators

One type of jet-flow agitator is shown in FIGS. 3-3A. This type of mixeris available commercially, e.g., from IKA under the tradename ROTOTRON™.Referring to FIG. 3, the mixer 200 includes a motor 202, which rotates adrive shaft 204. A mixing element 206 is mounted at the end of the driveshaft 204. As shown in FIG. 3A, the mixing element 206 includes a shroud208 and, within the shroud, an impeller 210. As indicated by the arrows,when the impeller is rotated in its “forward” direction, the impeller210 draws liquid in through the open upper end 212 of the shroud andforces the liquid out through the open lower end 214. Liquid exiting end214 is in the form of a high velocity stream or jet. If the direction ofrotation of the impeller 210 is reversed, liquid can be drawn in throughthe lower end 214 and ejected through the upper end 212. This can beused, for example, to suck in solids that are floating near or on thesurface of the liquid in a vessel. (It is noted that “upper” and “lower”refer to the orientation of the mixer in FIG. 3; the mixer may beoriented in a vessel so that the upper end is below the lower end.)

The shroud 208 includes flared areas 216 and 218 adjacent its ends.These flared areas are believed to contribute to the generally toroidalflow that is observed with this type of mixer. The geometry of theshroud and impeller also concentrate the flow into a high velocitystream using relatively low power consumption.

Preferably, the clearance between the shroud 208 and the impeller 210 issufficient so as to avoid excessive milling of the material as it passesthrough the shroud. For example, the clearance may be at least 10 timesthe average particle size of the solids in the mixture, preferably atleast 100 times.

In some implementations, the shaft 204 is configured to allow gasdelivery through the shaft. For example, the shaft 204 may include abore (not shown) through which gas is delivered, and one or moreorifices through which gas exits into the mixture. The orifices may bewithin the shroud 208, to enhance mixing, and/or at other locationsalong the length of the shaft 204.

The impeller 210 may have any desired geometry that will draw liquidthrough the shroud at a high velocity. The impeller is preferably amarine impeller, as shown in FIG. 3A, but may have a different design,for example, a Rushton impeller as shown in FIG. 3B, or a modifiedRushton impeller, e.g., tilted so as to provide some axial flow.

In order to generate the high velocity flow through the shroud, themotor 202 is preferably a high speed, high torque motor, e.g., capableof operating at 500 to 20,000 RPM, e.g., 3,000 to 10,000 RPM. However,the larger the mixer (e.g., the larger the shroud and/or the larger themotor) the lower the rotational speed can be. Thus, if a large mixer isused, such as a 5 hp, 10 hp, 20 hp, or 30 hp or greater, the motor maybe designed to operate at lower rotational speeds, e.g., less than 2000RPM, less than 1500 RPM, or even 500 RPM or less. The torque of themotor is preferably self-adjusting, to maintain a relatively constantimpeller speed as the mixing conditions change over time.

Advantageously, the mixer can be oriented at any desired angle orlocation in the vessel, to direct the jet flow in a desired direction.Moreover, as discussed above, depending on the direction of rotation ofthe impeller the mixer can be used to draw fluid from either end of theshroud.

In some implementations, two or more jet mixers are positioned in thevessel, with one or more being configured to jet fluid upward (“uppump”) and one or more being configured to jet fluid downward (“downpump”). In some cases, an up pumping mixer will be positioned adjacent adown pumping mixer, to enhance the turbulent flow created by the mixers.If desired, one or more mixers may be switched between upward flow anddownward flow during processing. It may be advantageous to switch all ormost of the mixers to up pumping mode to disperse material at the liquidsurface, as up pumping creates significant turbulence at the surface.

Suction Chamber Jet Mixers

Another type of jet mixer includes a primary nozzle that delivers apressurized fluid from a pump, a suction inlet adjacent the primarynozzle through which ambient fluid is drawn by the pressure drop betweenthe primary nozzle and the wider inlet, and a suction chamber extendingbetween the suction inlet and a secondary nozzle. A jet of high velocityfluid exits the secondary nozzle.

An example of this type of mixer is shown in FIG. 4. As shown, in mixer600 pressurized liquid from a pump (not shown) flows through an inletpassage 602 and exits through a primary nozzle 603. Ambient liquid isdrawn through a suction inlet 604 into suction chamber 606 by thepressure drop caused by the flow of pressurized liquid. The combinedflow exits from the suction chamber into the ambient liquid at highvelocity through secondary nozzle 608. Mixing occurs both in the suctionchamber and in the ambient liquid due to the jet action of the exitingjet of liquid.

The nozzle shown in FIG. 5, referred to as an eductor nozzle, operatesunder a similar principle. A nozzle embodying this design iscommercially available under the tradename TeeJet®. As shown, in nozzle700 pressurized liquid flows in through an inlet 702 and exits a primarynozzle 704, drawing ambient fluid in to the open end 706 of a diffuser708. The combined flow exits the opposite open end 710 of the diffuserat a circulation flow rate A+B that is the sum of the inlet flow rate Aand the flow rate B of the entrained ambient fluid.

Jet Aeration Type Mixers

Another type of jet mixing system that can be utilized is referred to inthe wastewater industry as “jet aeration mixing.” In the wastewaterindustry, these mixers are typically used to deliver a jet of apressurized air and liquid mixture, to provide aeration. However, in thepresent application in some cases the jet aeration type mixers areutilized without pressurized gas, as will be discussed below. Theprinciples of operation of jet aeration mixers will be initiallydescribed in the context of their use with pressurized gas, for clarity.

An eddy jet mixer, such as the mixer 800 shown in FIG. 7, includesmultiple jets 802 mounted in a radial pattern on a central hub 804. Theradial pattern of the jets uniformly distributes mixing energythroughout the vessel. The eddy jet mixer may be centrally positioned ina vessel, as shown, to provide toroidal flow about the center axis ofthe vessel. The eddy jet mixer may be mounted on piping 806, whichsupplies high velocity liquid to the eddy jet mixer. In someimplementations air is also supplied to the eddy jet mixer. The highvelocity liquid is delivered by a pump 808 which is positioned outsideof the vessel and which draws liquid in through an inlet 810 in the sidewall of the vessel.

FIGS. 8 and 9 show two types of nozzle configurations that are designedto mix a gas and a liquid stream and eject a high velocity jet. Thesenozzles are configured somewhat differently from the eddy jet mixershown in FIG. 7 but function in a similar manner. In the system 900shown in FIG. 8, a primary or motive fluid is directed through a liquidline 902 to inner nozzles 904 through which the liquid travels at highvelocity into a mixing area 906. A second fluid, e.g., a gas, such ascompressed air, nitrogen or carbon dioxide, or a liquid, enters themixing area through a second line 908 and entrained in the motive fluidentering the mixing area 906 through the inner nozzles. In someinstances the second fluid is nitrogen or carbon dioxide so as to reduceoxidation of the enzyme. The combined flow from the two lines is jettedinto the mixing vessel through the outer nozzles 910. If the secondfluid is a gas, tiny bubbles are entrained in the liquid in the mixture.Liquid is supplied to the liquid line 902 by a pump. Gas, if it is used,is provided by compressors. If a liquid is used as the second fluid, itcan have the same velocity as the liquid entering through the liquidline 902, or a different velocity.

FIG. 9 shows an alternate nozzle design 1000, in which outer nozzles1010 (of which only one is shown) are positioned along the length of anelongated member 1011 that includes a liquid line 1002 that ispositioned parallel to a second line 1008. Each nozzle includes a singleouter nozzle 1010 and a single inner nozzle 1004. Mixing of the motiveliquid with the second fluid proceeds in the same manner as in thesystem 900 described above.

In some embodiments, the jet nozzles are arranged to cause the contentsof the tank to both revolve and rotate in a toroidal, rolling donutconfiguration around a central vertical axis of the vessel. Flow aroundthe surface of the toroid is drawn down the vessel center, along thefloor, up the walls and back to the center, creating a rolling helixpattern, which sweeps the center and prevents solids from settling. Thetoroidal pattern is also effective in moving floating solids to thevessel center where they are pulled to the bottom and become homogenouswith the vessel contents. The result is a continuous helical flowpattern, which minimizes dead spots in the vessel contents.

Backflushing

In some instances, the jet nozzles described herein can become plugged,which may cause efficiency and cost effectiveness to be reduced.Plugging of the nozzles may be removed by reversing flow of the motiveliquid through the nozzle.

Other Mixing Systems Low Speed Agitators

FIGS. 10 and 10A illustrate agitators configured to be mounted on ashaft (not shown) for rotational mixing at relatively low speeds. Theagitators may include, for example, two mixing elements 1702 (FIG. 10),or three mixing elements (FIG. 10A), mounted on support arms 1701 abouta central mounting hub 1703 that is disposed to receive a shaft.

The mixing elements 1702 are in the form of truncated cones, each ofwhich has a first end 1704 and a second end 1706. The first end has across-section greater than the cross-section of the second end. Themixing elements are positioned such that the central axes of the mixingelements are disposed at an angle relative to a plane of rotation of themixing elements.

The agitator is rotated in a direction so that liquid flows in throughthe first end 1704 and out through the second end 1706 at a highervelocity, creating dynamic flow conditions by generating turbulent flowat the tapered end of each mixing element. The angulation of the mixingelements relative to the plane of rotation tends to cause a continuousclosed circular flow which in the vicinity of an adjacent vessel wallflows upwardly and in the central part of the vessel flows downwardlycoaxially to the mixer shaft where it passes through the intermediatespaces between the support arms 1701. The intensity of this circularflow depends on the magnitude of the angle.

Mixers of this type are available commercially from Inotec under thetradename Visco-Jet™.

Materials Medium

Bioprocessing, e.g., fermentation, is generally conducted in an aqueousgrowth medium, which can contain a nitrogen source or other nutrientsource, e.g., urea, along with vitamins and trace minerals and metals.It is generally preferable that the growth medium be sterile, or atleast have a low microbial load, e.g., bacterial count. Sterilization ofthe growth medium may be accomplished in any desired manner. However, inpreferred implementations, sterilization is accomplished by irradiatingthe growth medium or the individual components of the growth mediumprior to mixing. The dosage of radiation is generally as low as possiblewhile still obtaining adequate results, in order to minimize energyconsumption and resulting cost. For example, in many instances, thegrowth medium itself or components of the growth medium can be treatedwith a radiation dose of less than 5 Mrad, such as less than 4, 3, 2, or1 Mrad. In specific instances, the growth medium is treated with a doseof between about 1 and 3 Mrad.

If the pH of the vessel contents is too low, this may tend to inhibitfermentation with some microorganisms, such as Pichia stipitus.Accordingly, it is in some cases desirable to add base and/or a buffer,before or during fermentation, to bring up the pH of the solution. Forexample, sodium hydroxide or lime can be added to the fermentationmedium to elevate the pH of the medium to a range that is optimum forthe microorganism utilized.

An example of a bioprocessing medium has the following concentrations ofcomponents:

Yeast nitrogen base  1.7 g/L Urea 2.27 g/L Peptone 6.56 g/L Tween ® 80surfactant   10 g/L

Microorganisms

Bioprocesses can utilize various microorganisms.

The microorganism may be a cell, e.g., a eukaryotic or prokaryotic cell.Eukaryotic cells include animal (e.g., mammalian) cells, plant cells,fungal cells, glaucophytes, haptophytes, cryptomonads, and amoeboidprotists, e.g., amoebozoans and foraminifera. Prokaryotic cells includebacteria and archaea.

The microorganism can be a natural microorganism and/or an engineeredmicroorganism. For example, the microorganism can be a bacterium, e.g.,a cellulolytic bacterium, a fungus, e.g., a yeast, a plant or a protist,e.g., an algae, a protozoa or a fungus-like protist, e.g., a slime mold.When the organisms are compatible, mixtures of organisms can beutilized. The microorganism can be an aerobe or an anaerobe. Themicroorganism can be a homofermentative microorganism (produces a singleor a substantially single end product). The microorganism can be ahomoacetogenic microorganism, a homolactic microorganism, a propionicacid bacterium, a butyric acid bacterium, a succinic acid bacterium or a3-hydroxypropionic acid bacterium. The microorganism can be of a genusselected from the group consisting of Clostridium, Lactobacillus,Moorella, Thermoanaerobacter, Proprionibacterium, Proionispera,Anaerobiospirillum, and Bacteriods. In specific instances, themicroorganism can be Clostridium formicoaceticum, Clostridium butyricum,Moorella thermoacetica, Thermoanearobacter kivui, Lactobacillusdelbrukii, Propionibacterium acidipropionici, Propionispera arboris,Anaerobiospirillum succiniproducens, Bacteroides amylophilus, orBacteroides ruminicola. For example, the microorganism can be arecombinant microorganism engineered to produce a desired product, suchas recombinant Escherichia coli transformed with one or more genescapable of encoding proteins that direct the production of the desiredproduct (see, e.g., U.S. Pat. No. 6,852,517, issued Feb. 8, 2005).

Enzymes

Enzymes, such as cellobiases and cellulases, are used in somebioprocesses.

Cellobiases include a cellobiase from Aspergillus niger sold under thetradename NOVOZYME 188™.

A cellulase may be of fungal or bacterial origin. Cellulases includethose from the genera Bacillus, Pseudomonas, Humicola, Fusarium,Thielavia, Acremonium, Chrysosporium and Trichoderma, and includespecies of Humicola, Coprinus, Thielavia, Fusarium, Myceliophthora,Acremonium, Cephalosporium, Scytalidium, Penicillium or Aspergillus(see, e.g., EP 458162), especially those produced by a strain selectedfrom the species Humicola insolens (reclassified as Scytalidiumthermophilum, see, e.g., U.S. Pat. No. 4,435,307), Coprinus cinereus,Fusarium oxysporum, Myceliophthora thermophila, Meripilus giganteus,Thielavia terrestris, Acremonium sp., Acremonium persicinum, Acremoniumacremonium, Acremonium brachypenium, Acremonium dichromosporum,Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum,Acremonium incoloratum, and Acremonium furatum; preferably from thespecies Humicola insolens DSM 1800, Fusarium oxysporum DSM 2672,Myceliophthora thermophila CBS 117.65, Cephalosporium sp. RYM-202,Acremonium sp. CBS 478.94, Acremonium sp. CBS 265.95, Acremoniumpersicinum CBS 169.65, Acremonium acremonium AHU 9519, Cephalosporiumsp. CBS 535.71, Acremonium brachypenium CBS 866.73, Acremoniumdichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, Acremoniumpinkertoniae CBS 157.70, Acremonium roseogriseum CBS 134.56, Acremoniumincoloratum CBS 146.62, and Acremonium furatum CBS 299.70H. Cellulolyticenzymes may also be obtained from Chrysosporium, preferably a strain ofChrysosporium lucknowense. Additionally, Trichoderma (particularlyTrichoderma viride, Trichoderma reesei, and Trichoderma koningii),alkalophilic Bacillus (see, for example, U.S. Pat. No. 3,844,890 and EP458162), and Streptomyces (see, e.g., EP 458162) may be used.

Enzyme complexes may be utilized, such as those available from Genencoreunder the tradename ACCELLERASE®, for example, Accellerase® 1500 enzymecomplex. Accellerase 1500 enzyme complex contains multiple enzymeactivities, mainly exoglucanase, endoglucanase (2200-2800 CMC U/g),hemi-cellulase, and beta-glucosidase (525-775 pNPG U/g), and has a pH of4.6 to 5.0. The endoglucanase activity of the enzyme complex isexpressed in carboxymethylcellulose activity units (CMC U), while thebeta-glucosidase activity is reported in pNP-glucoside activity units(pNPG U). In one embodiment, a blend of Accellerase® 1500 enzyme complexand NOVOZYME™ 188 cellobiase is used.

Surfactants

The addition of surfactants can enhance the rate of some bioprocesses.Examples of surfactants include non-ionic surfactants, such as a Tween®20 or Tween® 80 polyethylene glycol surfactants, ionic surfactants, oramphoteric surfactants. Other suitable surfactants include octylphenolethoxylates such as the TRITON™ X series nonionic surfactantscommercially available from Dow Chemical. A surfactant can also be addedto keep a product that is being produced in solution, particularly inhigh concentration solutions.

Other Embodiments

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made without departing fromthe spirit and scope of the disclosure.

For example, For example, the jet mixers described herein can be used inany desired combination, and/or in combination with other types ofmixers. While a single jet mixer is shown in FIG. 1, a plurality of jetmixers can be used, e.g., as shown in FIG. 11, or a jet mixer could beused in combination with a different type of mixer, e.g., a marineimpeller mixer.

The jet mixer(s) may be mounted in any desired position within thevessel. With regard to shaft-mounted jet mixers such as the one shown inFIG. 1, the shaft may be collinear with the center axis of the vessel,or may be offset therefrom, e.g., as shown in FIG. 11. For example, ifdesired the vessel may be provided with a centrally mounted mixer of adifferent type, e.g., a marine impeller or Rushton impeller, and a jetmixer may be mounted in another area of the vessel either offset fromthe center axis or on the center axis. In the latter case one mixer canextend from the top of the vessel while the other extends upward fromthe floor of the vessel.

In any of the jet mixing systems described herein, the flow of fluid(liquid and/or gas) through the jet mixer can be continuous or pulsed,or a combination of periods of continuous flow with intervals of pulsedflow. When the flow is pulsed, pulsing can be regular or irregular. Inthe latter case, the motor that drives the fluid flow can be programmed,for example to provide pulsed flow at intervals to prevent mixing frombecoming “stuck.” The frequency of pulsed flow can be, for example, fromabout 0.5 Hz to about 10 Hz, e.g., about 0.5 Hz, 0.75 Hz, 1.0 Hz, 2.0Hz, 5 Hz, or 10 Hz. Pulsed flow can be provided by turning the motor onand off, and/or by providing a flow diverter that interrupts flow of thefluid.

The bioreactors described herein, or portions thereof, can be mobile,for example as described in U.S. Ser. No. 12/374,549, the fulldisclosure of which is incorporated herein by reference.

Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A bioreactor comprising a vessel, a jet mixingsystem configured to draw fluid contents from the vessel, through aninlet in a wall of the vessel to piping that is external to the vessel,and jet the fluid contents from the piping back into the vessel, a gasdelivery system configured to supply a process gas to the vessel, and atemperature control system configured to regulate temperature in thevessel.
 2. The bioreactor of claim 1 further comprising a plurality ofsensors.
 3. The bioreactor of claim 2 further comprising a processcontroller configured to receive input from the sensors.
 4. Thebioreactor of claim 1 further comprising delivery devices configured todeliver to the vessel a medium, microorganisms or cells, and/or othermaterials used in a bioprocess.
 5. The bioreactor of claim 1 wherein thevessel includes a vent, and the bioreactor includes a source of oxygenin communication with the vessel, an oxygen monitor configured tomonitor the oxygen content of a liquid in the vessel, and a controllerconfigured to adjust the oxygen content of the liquid, using the ventand oxygen source, in response to input from the oxygen monitor.
 6. Thebioreactor of claim 1 further comprising a concentration monitorconfigured to monitor the concentration of a product in the liquid inthe vessel, and a controller configured to stop a bioprocess takingplace within the vessel based on input received from the concentrationmonitor.
 7. The bioreactor of claim 1 wherein the vessel includes anoutlet through which effluent can be collected.
 8. The bioreactor ofclaim 1 wherein the jet mixing system is configured to limit anyincrease in the overall temperature of a fluid medium in the vessel toless than 5 degrees C. over the course of mixing.
 9. The bioreactor ofclaim 1 wherein the vessel is formed of glass and/or stainless steel.10. The bioreactor of claim 1 wherein the vessel is jacketed.
 11. Thebioreactor of claim 1 wherein the vessel includes a port through whichthe contents of the vessel can be harvested or sampled.
 12. Thebioreactor of claim 2 wherein said sensors include a foam sensor, adissolved oxygen sensor, and a pH sensor.
 13. The bioreactor of claim 1wherein the jet mixing system includes a torque controller.
 14. Thebioreactor of claim 1 wherein the jet mixing system is configured forpulsating flow.
 15. The bioreactor of claim 14 wherein the jet mixingsystem comprises a flow diverter.